Single-molecule experiments on protein and RNA folding promise unprecedented insights into the formation of molecular structure, its relationship to biological function, and diseases associated with folding defects. Such studies-like the manipulation of biomolecules in an optical tweezer force clamp-aim for a detailed, quantitative description of the folding free energy landscape and dynamics. Yet the raw data ac- cessible to the experimentalist is only an indirect measure of the protein/RNA configuration over time. The crucial theoretical challenge addressed by this research proposal is how to translate the data into a com- plete picture of the underlying folding landscape. Without a comprehensive solution to this problem there is no way to disentagle which aspects of the experimental trajectories are due to the object of interest, and which are artifacts of the measuring apparatus-in short, the full potential of single-molecule techniques cannot be realized. The proposal has the following specific goals: (1) For the case of an equilibrium optical tweezer force clamp system, devise a theoretical procedure to extract the full biomolecular free energy landscape as a function of extension, together with kinetic properties like folding rates. This will re- quire filtering out the influence of other system components- the attached DNA handles and polystyrene beads-as well as correcting for tension fluctuations and hydrodynamic interactions. (2) Implement the extraction method as an easy-to-use, open-source software package that will directly process the exper- imental time series data. The design and testing of this package will be carried out with experimental collaborators, who have recently measured optical tweezer trajectories for the folding/unfolding of a yeast leucine zipper protein domain. (3) To provide an interpretative framework and testable predictions for fu- ture force clamp experiments, run long-time, coarse-grained simulations of the T. Thermophila ribozyme in an optical tweezer. The object will be to relate the extracted free energy landscape to the precise role of counterions in the formation of the RNA tertiary structure-an aspect of the RNA folding problem that is still not quantitatively understood. Put together, the results of the proposed research will constitute a necessary step for the further progress of optical tweezer studies, and serve as a prototype for tackling measurement problems endemic in all single-molecule systems. PUBLIC HEALTH RELEVANCE: The molecular mechanisms of how proteins and RNAs fold are fundamental to understanding their roles in catalyzing biochemical reactions, and neurodegenerative diseases like Alzheimer's and Parkinson's related to misfolding. The proposed research uses theory and computation to interpret protein/RNA single-molecule experimental data, in order to extract a detailed picture of the folding energy landscape and dynamics.